The broad objectives of this research are to elucidate the functions of the centrosome in vertebrate somatic cells, and the mechanisms by which it accomplishes these functions. The proposed work is made possible by the successful development of a gamma-tubulin-GFP fusion protein that, for the first time, makes the centrosome clearly visible in living vertebrate somatic cells throughout the cell cycle. Aim number 1 is to clarify the role the centrosome in spindle formation and maintenance during mitosis in vertebrate somatic cells. To do this I will use laser microsurgery to specifically destroy one or both of the centrosomes at various stages of mitosis, from mid-prophase through anaphase. Aim number 2 is to determine if cells initiate and complete DNA synthesis, and/or undergo division, after destroying (by laser microsurgery) all or just part of the centrosome during different periods of cell cycle. These experiments will reveal whether the absence of a centrosome, or the presence of a centrosome containing just one centriole in G1 or a single diplosome (i.e., a mother/daughter centriole pair) in G2, inhibits normal progression through the cell cycle in vertebrate somatic cells. Aim number 3 is to determine the rates with which gamma-tubulin exchanges between the centrosome-associated fraction and the cytoplasmic pool during different periods of cell cycle in untreated cells, and when microtubule dynamics is altered by drugs that affect their stability (e.g., nocodazole, taxol). These experiments will reveal if the centrosome recruits gamma-tubulin only when it is needed (e.g., at the G2/M transition), or if it recruits gamma-tubulin continuously throughout the cell cycle but maintains part of it in an inactive form. This study will also provide additional information on whether the centrosome cycle continues to run in the absence of normal microtubule behavior. Together, the novel data obtained from these studies will allow researchers to better define how the centrosome is involved in cell proliferation and cell cycle control, and may lead to the development of new strategies for the treatment of disease states related to microtubule function (e.g., as cancer, Alzheimer's, arthritis, etc.)